Escherichia coli containing mutated lpdA gene and application thereof

Abstract

The invention relates to the field of modifying E. coli through genetic engineering. Specifically, the invention provides an E. coli containing a mutated lpdA gene. The invention also relates to use of the E. coli in the production of chemical material such as ethanol, and succinate etc. The invention also provides a method of producing chemical materials such as ethanol and succinate etc. by using the E. coli , as well as a method for increasing the activity of pyruvate dehydrogenase in E. coli by introducing a mutated lpdA gene.

The invention claimed is: 
     
       1. A recombinant  E. coli , wherein said  E. coli  contains a mutated lpdA gene encoding lipoamide dehydrogenase, wherein the polypeptide encoded by the mutated lpdA gene consists of the amino acid sequence SEQ ID No.: I with a modification at position A358 of SEQ ID No.: 1 and optionally modification(s) at one or both positions T81 and P275, SEQ ID No.: 1,
 wherein the modification at the position T81, is the replacement of T with I; the modification at the position P275 is the replacement of P with S; and the modification at the position A358 is the replacement of A with V; 
 optionally, the expression of the mutated lpdA gene, or the activity of the protein encoded by said mutated lpdA gene, in the recombinant  E. coli , is enhanced. 
 
     
     
       2. The  E. coli  of  claim 1 , wherein said mutated lpdA gene consists of the nucleotide sequence SEQ ID No.: 2 with a mutation at position C1073 of SEQ ID No.: 2 and optionally at one or both positions of the positions C242 and C823, of SEQ ID No.: 2,
 optionally the mutation is the replacement of C with T. 
 
     
     
       3. The  E. coli  of  claim 1 , wherein the polypeptide encoded by said mutated lpdA gene consists of the amino acid sequence SEQ ID No.: 1 with the modifications at the positions T81, P275 and A358 of SEQ ID No.: 1. 
     
     
       4. The  E. coli  of  claim 2 , wherein said mutated lpdA gene consists of the nucleotide sequence SEQ ID No.: 2 with the mutation at the positions C242, C823 and C1073 of SEQ ID No.: 2. 
     
     
       5. The  E. coli  of  claim 1 , wherein said mutated lpdA gene is in a plasmid or a chromosome. 
     
     
       6. The  E. coli  of  claim 1 , wherein said  E. coli  also contains the modifications of:
 inhibited expression of the gene(s) involved in phosphoenolpyruvate:sugar phosphotransferase system (PTS), or inhibited activities of the protein(s) encoded by the gene(s) involved in phosphoenolpyruvate:sugar phosphotransferase system (PTS); 
 inhibited expression of pflB or adhE genes, or inhibited activities of the protein(s) encoded by pflB and/or adhE genes; 
 inhibited expression of ldhA gene, or inhibited activity of the protein encoded by ldhA gene; 
 enhanced expression of galP gene or exogenous glf gene, or enhanced activities of the protein(s) encoded by galP gene or exogenous glf gene; and 
 enhanced expression of pck gene, or enhanced activity of the protein encoded by pck gene, 
 wherein the pflB encodes pyruvate formate lyase, the adhE gene encodes ethanol/acetaldehyde dehydrogenase, the ldhA gene encodes lactate dehydrogenase, the galP gene encodes galactose MFS transporter, the glf gene encodes glucose transporter Glf glucose facilitator protein), and the pck gene encodes phosphenolpyruvate carboxykinase. 
 
     
     
       7. The  E. coli  of  claim 6 , wherein said gene(s) involved in phosphoenolpyruvate:sugar phosphotransferase system (PTS) are one or more genes selected from the group consisting of genes ptsI encoding PTS system enzyme I, ptsH encoding PTS system enzyme Hpr, crr encoding PTS system enzyme IIA Glc  and ptsG encoding PTS system enzyme IICB Glc . 
     
     
       8. The  E. coli  of  claim 1 , wherein said  E. coli  also contains the genetic modifications of:
 inhibited expression of pflB gene, or inhibited activity of the protein encoded by pflB gene; 
 inhibited expression of ldhA gene, or inhibited activity of the protein encoded by ldhA gene; and 
 inhibited expression of frdABCD gene cluster, or inhibited activities of the protein(s) encoded by frdABCD gene cluster, 
 wherein the frdABCD gene cluster encodes fumarate reductase, including frdA gene encoding fumarate reductase flavoprotein subunit, frdB gene encoding fumarate reductase iron-sulphur protein subunit, frdC gene encoding fumarate reductase subunit C and frdD gene encoding fumarate reductase subunit D. 
 
     
     
       9. The  E. coli  of  claim 1 , wherein said  E. coli  also contains the genetic modification of: enhanced expression of aceEF gene cluster, or enhanced activities of the protein(s) encoded by aceEF gene cluster, wherein the aceEF gene cluster encodes pyruvate complex E1/E2, including aceE gene encoding pyruvate dehydrogenase complex E1 and aceF gene encoding pyruvate dehydrogenase complex E2. 
     
     
       10. A method for producing ethanol and/or succinate, comprising culturing the  E. coli  of  claim 1 .